Age-Related Changes in Aortic Valve Hemostatic Protein Regulation
Objective—Although valvular endothelial cells have unique responses compared with vascular endothelial cells, valvular regulation of hemostasis is not well-understood. Heart valves remodel throughout a person’s lifetime, resulting in changes in extracellular matrix composition and tissue mechanical properties that may affect valvular endothelial cell hemostatic function. This work assessed valvular endothelial cell regulation of hemostasis in situ and in vitro as a function of specimen age.
Approach and Results—Porcine aortic valves were assigned to 1 of 3 age groups: Young (YNG) (6 weeks); Adult (ADT) (6 months); or Elderly (OLD) (2 years). Histological examination of valves showed that secreted thrombotic/antithrombotic proteins localize at the valve endothelium and tissue interior. Gene expression and immunostains for von Willebrand factor (VWF), tissue factor pathway inhibitor, and tissue plasminogen activator in YNG porcine aortic valve endothelial cells were higher than they were for OLD, whereas plasminogen activator inhibitor 1 levels in OLD were higher than those for YNG and ADT. Histamine-stimulated YNG porcine aortic valve endothelial cells released higher concentrations of VWF proteins than OLD, and the fractions of VWF-140 fragments was not different between age groups. A calcific aortic valve disease in vitro model using valvular interstitial cells confirmed that VWF in culture significantly increased valvular interstitial cell nodule formation and calcification.
Conclusions—Hemostatic protein regulation in aortic valve tissues and in valvular endothelial cells changes with age. The presence of VWF and other potential hemostatic proteins increase valvular interstitial cell calcification in vitro. Therefore, the increased capacity of elderly valves to sequester the hemostatic proteins, together with age-associated loss of extracellular matrix organization, warrants investigation into potential role of these proteins in the formation of calcific nodules.
Semilunar valve diseases, particularly those affecting the aortic valve (AV), cause >60% of valve disease mortality, and ≈50 000 procedures to replace or repair semilunar valves are performed each year in the United States.1 Semilunar valve dysfunction affects all ages, from congenital valve defects experienced by neonates and children to the growing number of calcified valves in the elderly.1–6 These dysfunctional heart valves most often require surgical replacement using mechanical or bioprosthetic valves that may fail over time because of structural or thrombosis-related problems.6,7
To understand valvular disease and develop noninvasive therapeutic solutions, it is necessary to improve fundamental knowledge about the response of valve cells and extracellular matrix (ECM) to surrounding environments in various physiological states. The characterization of changes in valvular biology with respect to aging has become especially important as the occurrence of AV sclerosis and calcification continues to increase in the elderly population.4,5,8
Although most studies of valvular biology use AV tissues and cells from adults (either animal models or human specimens resected at surgery), it is clear that consideration of specimen age is necessary to evaluate age-specific conditions and pathologies. Previous studies from our research group and others have identified numerous age-related changes in the ECM composition and mechanical properties of the AV, as well as in the valve cell phenotypes.6,9–12 Considerable tissue remodeling and growth occurs in AV tissues before adulthood.4,11,13 During fetal development, AV tissues have not yet formed trilaminate ECM structures or high degree of elastin and collagen alignment that is apparent in adult valves.6,11,13 To mediate this microstructural organization of the mature AV, the developing AV also demonstrates cellular activation of both valvular interstitial cells and valvular endothelial cells (VECs).6,11,12
As subjects reach adulthood, valve cells become quiescent and maintain homeostasis of the highly organized valve leaflet structure and function.6 However, the AV continues to change with aging. Older valves have increased thickness and increased levels of collagen type III interspersed with proteoglycans and glycosaminoglycans throughout the leaflets.10,14–16 Corresponding to the altered ECM organization in older valves, the stiffness in the circumferential direction increases with age as well.10 Several studies have suggested that the remodeling occurring in elderly AV tissues is mediated by the activation of valvular interstitial cells, which can lead to osteogenic differentiation and ultimately valve calcification.2,6,10,17 However, additional factors including VEC dysfunction, changes in hemodynamics and valve mechanics, and ECM remodeling may also influence calcific formation in valve tissues. Thus, as valves remodel throughout a lifetime, the resulting changes in ECM composition and tissue mechanical properties are likely to affect cellular behavior and increase the risk of valvular diseases.
VECs have been identified to play an important role in maintaining valve homeostasis as well as to affect the onset of valvular disease. However, many aspects of VEC function and behavior remain unknown. Recent work has shown that VECs have distinct phenotypes compared with vascular and cardiac endothelial cells, for example, VECs align perpendicular to the direction of fluid flow, unlike vascular endothelial cells, and have different mechanotransduction pathways and gene expression, likely attributable to the unique flow and mechanics to which VECs are subjected.18–20 VECs have been observed to have strong interactions with valvular interstitial cells and ECM and seem to be highly sensitive to their surrounding environments.19,21,22 These cells can undergo endothelial to mesenchymal transdifferentiation, and thus play an important role in valvulogenesis during fetal development.23,24 Furthermore, previous studies have suggested that VEC dysfunction may play a role in initiating valvular diseases.24–27
Despite these links between VECs and valve diseases, little has been accomplished to characterize the fundamental hemostatic behavior of VECs and the relation between this behavior and the valvular ECM in health, disease, and aging conditions. It is well-known that vascular endothelial cells perform substantial antithrombotic roles via production and release of tissue plasminogen activator (tPA), tissue factor pathway inhibitor (TFPI), von Willebrand factor (VWF) cleaving enzyme (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13 [ADAMTS-13]), nitric oxide, and heparin.28–30 Endothelial cells are also responsible for the production and release of thrombotic protein mediators such as plasminogen activator inhibitor 1 (PAI-1), tissue factor (TF), VWF, and P-selectin.28–30 Under homeostatic conditions, vascular endothelial cells constitutively express and produce both antithrombotic and thrombotic mediators to balance hemostatic effects and prevent thrombus formation. However, blood coagulation and thrombus formation are quickly initiated by endothelial cells in response to mechanical damage, ECM degradation, and disease.28–30 The production and regulation of these hemostatic factors have not been well-characterized in VECs.
This study will evaluate the aging-related changes in antithrombotic and thrombotic capacity of AV tissues and VECs. We hypothesize that aging affects the VEC regulation of hemostatic proteins and their interaction with valve ECM components, resulting in an unbalanced regulation of antithrombotic and thrombotic proteins within the tissues. Thus, the evaluation of AV tissues and VEC function with respect to antithrombotic and thrombotic functions in aging valves will provide insight into the specific factors and conditions that affect VEC hemostatic regulation. This information is also relevant to the development of valve replacement devices because the breakdown and thrombosis formation associated with mechanical and decellularized valve implants have generated interest in understanding how the natural antithrombotic behavior of VECs can be integrated into age-specific tissue-engineered heart valves. Overall, there is a critical need to understand the hemostatic behavior of VECs.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Hemostatic Proteins Distribute Differently Within AV Tissues
Immunostaining showed that endothelial cell–mediated antithrombotic and thrombotic proteins were present in each of the differently aged AV leaflet groups (Figure 1). The proteins were often localized with the endothelium at the edges of each leaflet. Many of these hemostatic mediators are actually secreted by endothelial cells and correspondingly were also found within the tissue interior to varying degrees.30 To confirm the subendothelial localization of ADAMTS-13 and VWF, immunostains were repeated on elderly, 2-year-old porcine aortic valve sample (OLD) tissues using new antibodies specific for the proteins (Figure III in the online-only Data Supplement). These additional immunostains showed the same pattern.
There were no significant differences in the proportions of tissues stained between these proteins within young, 6-week-old porcine aortic valve (YNG) and OLD samples. The proportion of VWF in adult, 6-month-old porcine aortic valve samples (ADT) was significantly higher than the proportions of all other proteins except for ADAMTS-13 (Figure 2). However, the proportion of tissue stained for VWF was significantly higher in OLD tissues than in YNG tissues (P<0.05). There were trends of higher proportions of OLD valve tissues stained for PAI-1 (P=0.06) and TFPI (P=0.07) than in the corresponding portions of staining of YNG tissues, and a trend of higher proportion of TFPI staining in OLD tissues than in ADT tissues as well (P=0.09). The proportions of the valve tissues stained for PAI-1, TF, and tPA were low (<10%) among all age groups in comparison to the other proteins evaluated (≤40%; Figure 2), and these less abundant proteins were localized primarily in the endothelium (Figure 1).
Porcine Aortic Valve Endothelial Cell Hemostatic Protein Gene Regulation Changes With Age
Gene expression of the antithrombotic and thrombotic proteins was quantified with quantitative real-time polymerase chain reaction (Figure 3). Porcine aortic valve endothelial cell (PAVEC) gene expression in all age groups was quantified relative to porcine pulmonary artery endothelial cells (PPAECs; denoted by dotted lines in Figure 3). Gene expression for VWF in YNG PAVECs was significantly higher than in OLD PAVECs (48×; P<0.005). Similarly, gene expression for the antithrombotic proteins TFPI and tPA was 3-fold higher in YNG PAVECs than in OLD PAVECs (P<0.05 for each). Compared with the PPAEC control group, the YNG PAVEC gene expression was significantly higher for VWF (58×; P<0.0005), TFPI (3×; P<0.05), and tPA (30×; P<0.05).
There were no significant differences between ADT PAVECs and the other 2 age groups for all proteins. However, ADT PAVECs did have significantly higher gene expression of TFPI (2.5×; P<0.05) compared with the PPAEC controls.
The only protein that had the highest expression in the OLD PAVECs was the thrombotic protein PAI-1, which was 2-fold higher than in both YNG and ADT PAVECs (P<0.05). There were no significant differences in the gene expression for ADAMTS-13 and TF between the PAVEC groups and PPAEC controls. Furthermore, all PAVEC and PPAEC groups had ≈1000× higher gene expression levels for all hemostatic proteins than did human umbilical vein endothelial cells (HUVECs; data not shown).
PAVECs Synthesize Hemostatic Mediators In Vitro
Primary PAVECs harvested from differently aged AV tissues maintained their endothelial phenotype and stained positively for CD31 (data not shown). ADAMTS-13 and VWF were generally colocalized in all age groups, although there was a greater abundance of VWF staining overall (Figure 4, first column). However, VWF levels in the OLD PAVECs appeared more profuse than in the YNG PAVEC cultures.
PAVECs positively stained for low levels of TFPI and TF (Figure 4, second column). TFPI staining in YNG and ADT PAVECs was primarily localized intracellularly with some punctate staining outside of the cells; TFPI staining in OLD PAVECs was low, with punctate staining outside of the cells. For the YNG PAVEC cultures, there seemed to be higher levels of TFPI than of TF. Conversely, TF was more abundant than TFPI in OLD cultures.
PAI-1 and tPA were also detected in PAVEC cultures (Figure 4, third column). There was intracellular staining for PAI-1 in all PAVEC age groups, although punctate extracellular staining of PAI-1 was present in the OLD PAVEC cultures. Staining for tPA appeared to be more abundant in YNG and ADT PAVEC cultures than in OLD PAVEC cultures. Although the stains for PAI-1 and tPA were generally less abundant in the OLD PAVEC cultures, the complimentary proteins seemed to be colocalized with slightly higher levels of PAI-1.
PAVECs Can Mediate Functional VWF Release and Cleavage
Histamine stimulation of PAVECs in vitro initiated the rapid release of thrombogenic VWF multimer proteins into the culture medium. For the comparison of PAVEC age groups, VWF protein release paralleled the VWF gene expression trend in that YNG PAVECs released a significantly higher concentration of VWF protein than did OLD PAVECs (P<0.05; Figure 5A). VWF release by the PPAECs and ADT PAVECs was not significantly different from that released by the YNG and OLD PAVECs. HUVECs released significantly higher amounts of VWF protein than all porcine-derived PAVEC and PPAEC groups (P<0.001).
The fraction of cleaved VWF-140 fragments was measured using enzyme-linked immunosorbent assays with a specific monoclonal antibody for cleaved VWF proteins. There was no statistically significant difference in the fraction of VWF-140 fragments between porcine-derived PAVECs and PPAECs (Figure 5B). For the HUVECs, only ≈10% of the VWF protein was cleaved into VWF-140 fragments, which was a significantly lower cleavage product fraction compared with the OLD PAVECs (P<0.05).
PAVEC-Released VWF Increases Porcine Aortic Valve Interstitial Cell Calcific Nodule Formation In Vitro
Porcine aortic valve interstitial cells (PAVICs) were cultured with various culture medium conditions to assess how the addition of VEC-released VWF affect calcific nodule formation in vitro. Measured levels of VWF protein present in the low serum PAVIC medium mixed with 3% (v/v) conditioned culture mediums collected from YNG, ADT, and OLD PAVEC groups were 257, 130, and 90 pg/mL, respectively. No levels of VWF protein were detected in any of the 3 control conditions.
Within 5 days of culture, nodule formation was visible in PAVIC cultures treated with each of the conditioned mediums from the differently aged PAVECs. At the end of the 10-day culture period, there were no significant differences in average number of nodules per well, average nodule size, or total calcified area between the control groups of low serum PAVIC medium, 3% PAVEC stimulation medium without histamine, and 3% PAVEC stimulation medium with histamine (Figure 6 and Figure VI in the online-only Data Supplement). The total calcified area in PAVICs treated with the conditioned mediums from YNG, ADT, and OLD PAVEC supernatants (2.0–2.4×106 µm2) were all significantly greater than all 3 control groups (1.2–3.2×104 µm2; P<0.001; Figure 6). PAVICs in both YNG (40 nodules) and ADT (45 nodules) conditioned medium groups had significantly more nodules per well than the 3 control groups (9–13 nodules; P<0.001; Figure VIA in the online-only Data Supplement). Furthermore, the average nodule size in PAVICs treated with 1 of the 3 conditioned mediums (52–78×103 µm2) were significantly larger than each of the 3 control conditions (13–23×103 µm2; P<0.001; Figure VIB in the online-only Data Supplement).
The endothelial cell–mediated process of hemostasis is essential for the function of all living heart valve tissues. As these tissues undergo remodeling with age and disease, VEC management of hemostatic protein regulation also changes. This study is the first to examine the production and expression of numerous hemostatic proteins in AV tissues and in vitro PAVEC cultures from 3 distinct age groups.
There are several age-related differences in the abundance and localization of thrombotic and antithrombotic proteins within the AV. As expected, all examined hemostatic proteins were strongly present at the leaflet edges in each age group. However, many of these soluble components, namely ADAMTS-13, TFPI, VWF, TF, and PAI-1, were found throughout the interior of the valves as well. These proteins were primarily localized in the ventricularis layer of the AV in YNG and ADT tissues. Conversely, they were distributed more evenly across the valve layers in OLD AV samples. Because of their interior location, it is possible that these hemostatic components interact with subendothelial ECM components such as elastin and collagen type I. The ventricularis layer contains densely packed elastin, which may sequester the soluble hemostatic proteins that have permeated into the subendothelium. As elastin becomes more disperse throughout the OLD AV tissues,10 the hemostatic proteins may be able to permeate throughout the entire interior of the AV.
These proteins may also interact with collagen type I because it is the most abundant valve ECM protein and is present throughout the leaflet. Previous work investigating fiber alignment in articular cartilage tissues suggests that highly organized collagen alignment limits diffusion of proteins through anisotropic tissues.31,32 Similarly, the lower amount of hemostatic proteins within the fibrosa layer of the YNG and ADT AV tissues may be attributable to the highly organized and aligned collagen fibrils serving as a barrier to limit the diffusion of hemostatic proteins into the aortic side of the AV leaflet. Given the age-associated collagen remodeling and turnover observed in older tissues,14 this barrier function of the fibrosa may be attenuated with age and may result in the increased permeation of VEC-secreted hemostatic proteins throughout the valve layers.
In addition, quantitative real-time polymerase chain reaction analyses showed highly significant changes in gene expression of many hemostatic proteins with age in PAVEC cultures. PAI-1, the protein inhibitor for tPA in the fibrinolysis pathways, was significantly elevated with age, as shown by mRNA analysis, immunocytochemistry, and a trend in the proportion of tissue stained. The increased accumulation of PAI-1 in older valves suggests that this smaller-molecular-weight protein can easily permeate through the remodeled ECM architectures in OLD valves. Previous studies have shown that inflammatory or cytokine stimuli can increase endothelial secretion of PAI-1 while not affecting tPA secretion.30,33 It seems likely that elderly patients with higher levels of inflammation, cholesterol, and tissue remodeling will also experience more procoagulant states in which PAI-1 levels in the blood and ECM overcome the basally secreted levels of tPA.33–35 Interestingly, there were no differences in tPA accumulation in AV tissues despite the significant decline in tPA gene expression in OLD PAVECs relative to YNG PAVECs. This may suggest that the expressed tPA is released more into the bloodstream as opposed to accumulating within valve tissues. The reduction in PAVEC tPA expression also reflects the hemostatic imbalance between tPA and PAI-1 associated with age.
PAVEC expression and tissue accumulation of TFPI and VWF were affected by age-related changes as well. In both cases, YNG PAVECs had significantly higher levels of TFPI and VWF gene expression than did OLD PAVECs. Consistent with quantitative real-time polymerase chain reaction results, stains for TFPI in YNG PAVECs appeared to be more abundant than in OLD PAVEC cultures. Conversely, OLD PAVEC cultures had slightly higher levels of VWF staining than in YNG PAVEC cultures. However, the polyclonal anti-VWF antibody used in the stain binds to both cleaved and uncleaved VWF proteins. Therefore, the increased staining observed in the OLD PAVEC cultures is likely from the higher proportion of cleaved VWF proteins in the condition, as observed in the enzyme-linked immunosorbent assay experiments. Furthermore, YNG valves showed trends of lower proportions of tissue stained for both TFPI and VWF than in OLD tissues. This discrepancy could be a result of the retention of these basally secreted proteins within the disorganized OLD tissue ECM through interactions with heparin and collagen type I.30,36
PAVEC expression of ADAMTS-13 and TF was not affected by age for gene expression or the proportion of tissue stained. Thus, these specific hemostatic proteins seem less likely to be direct players in causing age-related valve disease. These results, however, suggest that the valvular hemostasis may become unbalanced over a lifetime because many of the associated counteracting hemostatic proteins (VWF for ADAMTS-13, TFPI for TF) were shown to be affected by age. This imbalance was also observed in the immunostaining of PAVECs because colocalization of the associated hemostatic component pairs was less present in the OLD PAVEC cultures.
Functional testing of the fragmentation of histamine- stimulated VWF confirmed that PAVECs secrete ADAMTS-13 to cleave VWF multimers. The total amount of VWF protein released by OLD PAVECs was significantly lower than that released by YNG PAVECs, which was consistent with the gene expression results. The fractions of cleaved VWF (140 kDa fragments) were not significantly different between all porcine-derived endothelial cell groups, which supports the previous finding that ADAMTS-13 expression and function is not affected by age.37 On the contrary, because VWF expression and release are affected by age, and with the elderly age group having a decline in VWF expression and production, there will be lower proportions of complete VWF proteins present in patient plasma that can mediate coagulation. This phenomenon is consistent with clinical reports that elderly patients require lower doses of anticoagulants after heart valve surgeries than do adult patients because of lower clearance kinetics of the drugs and decreased clotting ability in elderly patients.38
The changes in hemostatic protein regulation by VECs from our results match the age-related hemostatic protein plasma levels previously reported.33–35,38 We think that these age-related imbalances in hemostatic protein regulation and production by VECs are not likely to cause spontaneous development of thrombus. However, when injury or insult occur at the valve endothelium, the aging-induced imbalance in the expression of antithrombotic and thrombotic proteins by VECs will lower the capability of the endothelium to maintain hemostasis and thus cause AV tissues to be more susceptible to thrombotic complications. Also, injury to the endothelium exposes the underlying tissue, and therefore the accumulation of these proteins in valve tissues along with the structural changes can induce further thrombotic events.
Aside from intravascular complications, accumulation of these proteins in valve tissues along with the structural changes can cause tissue-level progression of age-related diseases such as calcific aortic valve disease (CAVD). Using an in vitro CAVD model, PAVIC nodule formation experiments showed that the presence of VEC-released VWF significantly increased the total number of nodules, nodule area, and total calcified area in PAVICs relative to control groups. Having no differences in nodule formation between the 3 control culture conditions confirmed that the significant increase in calcific nodule formation by PAVICs was not because of the addition of PAVEC stimulation medium or residual levels of histamine in solution, but rather because of the presence of VEC-released proteins including VWF after histamine stimulation. Therefore, the secretion and accumulation of VWF and other hemostatic proteins within the valve tissues may affect the development and progression of CAVD and other acquired valvular diseases.
Although there were no significant differences in PAVIC nodule formation between the differently aged PAVEC-based conditions, the significant increases in VWF present within OLD valve tissues in conjunction with age-associated ECM disorganization seen in the histology slides suggest that calcification could result from increased pro-osteogenic VWF–valvular interstitial cell interactions, which correlated with the classical clinical presentation of CAVD in the elderly. Although VWF is the main component of histamine-stimulated PAVEC supernatant, other factors in the supernatant (eg, prostacyclin, platelet-activating factor, angiopoietin-2, and interleukin-8) may also intensify PAVIC nodule formation, and further investigation into how other hemostatic proteins within valve tissues can affect AV calcification is warranted.39–41 Nonetheless, our studies have shown that PAVECs will constitutively secrete VWF and other hemostatic factors, which will result in their accumulation of these proteins within the valve subendothelium. As ECM organization changes with age and the valve hemostatic protein regulation becomes more imbalanced, there is the potential that VWF and some of these proteins may play a substantial role in the progression of age-related valve disease.
This study was also the first to perform an extensive characterization of PAVEC hemostatic protein expression and production relative to vascular endothelial cell types PPAECs and HUVECs. PPAECs have been documented to produce significant levels of VWF and were chosen as a porcine vascular endothelial cell control group.42 Previous findings have found that porcine endothelial cultures isolated from the thoracic aorta expressed low levels of VWF mRNA and did not contain Weibel–Palade bodies, indicating that porcine endothelial cells have different regional capacities for hemostatic regulation.42,43 The gene expression of TFPI in YNG and ADT PAVECs and of tPA and VWF in YNG PAVECs were significantly higher than in the baseline PPAECs, suggesting that PAVECs have distinct hemostatic regulation activity. Gene expression levels differed greatly between PAVECs and HUVECs, with expression of the investigated proteins being ≈3 orders of magnitude higher in the PAVECs. HUVECs have been well-documented to produce and release VWF in vitro30,44 and thus are frequently used in endothelial cell studies and hemostatic experiments. However, the concentration of VWF protein released from histamine-stimulated HUVECs was significantly higher than all porcine-derived endothelial cell groups, suggesting that HUVECs are capable of storing more VWF in their Weibel–Palade bodies compared with porcine endothelial cells. Therefore, future studies regarding PAVEC expression and mediated protein functions should consider using porcine-derived vascular endothelial cells as baseline controls.
Limitations of this study include the inherent variability in the semiquantitative analysis of immunohistochemistry, which reduces the ability to distinguish between age groups. A larger sample size may help in confirming the promising trends. Still, this study examined several key endothelial cell–mediated hemostatic proteins. Additional factors such as ectonucleotidases, prostacyclin, and thrombomodulin are yet to be studied with respect to PAVEC hemostatic regulation. Future studies using a gene array for a wider panel of thrombotic and antithrombotic proteins in conjunction with ECM components and inflammatory markers may be informative in elucidating the relationship between the VECs regulation of their environment and how the protein accumulation influences the biology and pathology of the AV. Furthermore, although the interaction and balance of thrombotic and antithrombotic proteins are well understood at the apical side of the endothelium, further studies to investigate the interactions of each protein with subendothelial ECM and cells may be important in understanding the mechanisms that can promote the onset of valvular dysfunction. Finally, further investigation into the molecular mechanisms behind the age-related imbalances in PAVEC hemostatic protein regulation will greatly enhance our knowledge of these changes and may provide potential targets for the prevention and treatment of acquired valve abnormalities.
In conclusion, it is well-understood that valve tissues experience different environments with respect to age as a result of changes in valve mechanics, hemodynamics, and matrix composition. This study has identified age-related differences in PAVEC hemostatic protein regulation and the ability of basally secreted proteins to aggregate within differently aged AV tissues. In addition, we have verified that PAVECs express many of the same thrombotic and antithrombotic proteins as vascular endothelial cells. Although there was not one pattern for all hemostatic protein expression and aggregation with respect to age, we demonstrated age-related differences in the overall expression and localization of tPA, PAI-1, TFPI, and VWF in AV tissues and cells. Although thrombosis does not commonly occur as an age-associated dysfunction in valve tissues, we observed that thickened elderly valve tissues with ubiquitous distributions of elastin and collagen sequester high levels of the hemostatic proteins in the subendothelium, which warrants further investigation into potential roles for these aggregated proteins in the formation of calcific nodules that are so prevalent in older AVs.5,6 Our in vitro PAVIC studies suggest that the presence of these VWF promotes PAVIC calcification and that VWF and other hemostatic proteins within the tissue interior may have a potential role in the development of CAVD and other acquired abnormalities as age-related valve tissue ECM remodeling occurs. Thus, future studies regarding valve biology, pathology, and tissue engineering require consideration of environments that reflect the specific age group in question, including the necessary hemostatic framework.
We thank Dr Joel L. Moake for extensive project support including discussion of the data and manuscript. We thankfully acknowledge Nancy Turner and Leticia Nolasco for providing harvested primary HUVECs and guidance in conducting VWF release and cleavage assays. We also thank Adam Lin for his thoughtful discussion of the study results.
Sources of Funding
This research was funded by a predoctoral fellowship from the American Heart Association (to L.R. Balaoing), the National Institutes of Health (HL110063), the Mary R. Gibson Foundation, and the Mabel and Everett Hinkson Memorial Fund.
Valvular endothelial cells (VECs) play an important role in maintaining valve homeostasis and affect the onset of valve disease. However, little has been done to characterize the fundamental hemostatic behavior of VECs and its relation to the extracellular matrix in health, disease, and aging. This study was the first to perform an extensive age-dependent characterization for hemostatic proteins in valves and VEC hemostatic regulation relative to vascular endothelial cell types. Results identified age-related differences in VEC hemostatic protein regulation, and the increased capacity of specific proteins to aggregate within regions of elderly valves have been shown to have age-associated loss of extracellular matrix organization. In vitro calcific aortic valve disease model studies show that the presence of von Willebrand factor significantly increases valvular interstitial cell formation of calcific nodules relative to baseline controls. Therefore, the hemostasis imbalance with aging and the accumulations of hemostatic proteins may contribute to the formation of calcific nodules and warrants further investigation to determine the connection between VEC hemostatic mechanisms and the progression of valve disease. Furthermore, this works proposes the importance of selecting appropriate age- and species-based controls in future VEC and hemostatic work.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301936/-/DC1.
- Nonstandard Abbreviations and Acronyms
- a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13
- 6-month-old porcine aortic valve sample
- aortic valve
- calcific aortic valve disease
- extracellular matrix
- human umbilical vein endothelial cell
- 2-year-old porcine aortic valve sample
- plasminogen activator inhibitor 1
- porcine aortic valve endothelial cell
- porcine aortic valve interstitial cell
- porcine pulmonary artery endothelial cell
- tissue factor
- tissue factor pathway inhibitor
- tissue plasminogen activator
- valvular endothelial cell
- von Willebrand factor
- 6-week-old porcine aortic valve sample
- Received May 3, 2013.
- Accepted October 16, 2013.
- © 2013 American Heart Association, Inc.
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